Spontaneous mutations are produced by a number of mechanisms. Recent molecular studies of spontaneous mutagenesis have focused primarily on errors produced during DNA polymerization, the role of DNA damage on polymerization fidelity and the repair of spontaneous DNA lesions. Although roles for recombination- associated errors in spontaneous mutagenesis have often been postulated, studies of these roles have generally focused on mutations involving repeated DNA sequences or chromosomal rearrangements, postulating recombination-associated DNA misalignment. This proposal describes a novel association between recombination and spontaneous mutagenesis. Recombination-associated spontaneous 1bp deletion frameshift mutations were produced at very high frequencies at specific sites in the vicinity of mismatches carried on two separate bacteriophage T4 DNAs during their co-infection of Escherichia coli cells. The DNA sequences of the co-infecting phage differed by 1bp each at two closely-linked sites. The spontaneous frameshifts arose at hotspots near to, but not at, the mismatched sites at frequencies of approximately 10/5. This mutation frequency is an enormous increase ( greater than 10/4-fold) over the frequency of frameshifts at the same sites when E. coli cells are infected by either phage without the other. Studies with different pairs of phage sequences show that hotspot mutagenesis is not limited to a specific pair of mismatches. Despite the very high frequency of specific frameshifts, discovery of mutagenesis was initially serendipitous because the frequency is not high relative to typical recombination frequencies between DNA mismatches in T4 (greater than or equal to 10/3 when markers separated by greater than or equal to 10bp). The mutants are easily distinguished from recombinants by sequencing and their high frequency at specific hotspot sites makes the system ideal for both genetic and molecular studies designed to define the mutational mechanism(s) responsible. The proposal relies on using both the frequency and the DNA sequences of the hotspot mutations as clues to the role(s) of local DNA sequences and enzymes of DNA metabolism on mutagenesis. The goal is the development of a mutagenesis model that describes the DNA substrates and enzymes that participate in mutagenesis with enough precision that DNA sequence changes and mutant enzymes can be shown to produce predictable effects. Enzymatically detailed descriptions make the mechanism exportable to other genetic systems because variation expected from system-specific enzymology differences can be accommodated. The discovery of T4 recombination- associated spontaneous mutants in phage that are non-recombinant for the mismatched sequences that led to mutagenesis provides a new way to think how spontaneous mutagenesis may be promoted by the cryptic participation of other DNA sequences. For example, spontaneous mutagenesis may be higher in heterozygotes at highly polymorphic sites. Human disease alleles associated with nearby polymorphic repeat sequences and hypervariable regions in genes of the Major Histocompatibility Complex may be examples where the influence of local mismatches on nearby spontaneous mutagenesis could be substantial.